Alcohol abuse and alcohol dependence (i.e., alcoholism) are serious public health problems of modern society. In the United States alone, an estimated 13 million adults exhibit symptoms of alcohol dependence due to excessive alcohol intake, and an additional 7 million abuse alcohol without showing symptoms of dependence according to U.S. Government projections from studies conducted in the mid-1980s. Alcohol dependence and abuse are very expensive: in economic and medical terms, it will cost the U.S. well over $200 billion in 1991 with no prospect of falling or leveling off. The social and psychological damages inflicted on individuals as a consequence of alcohol abuse, e.g., children born with fetal alcohol syndrome (FAS) and victims of alcohol-related accidental death, homicide, suicide, etc., are immense.
While it is generally accepted that alcoholism and alcohol abuse are affliations with staggering international economic, social, medical, and psychological repercussions, success in preventing or otherwise ameliorating the consequences of these problems has been an elusive goal. Only very recently the public view that alcoholism and alcohol abuse are remeciable solely by moral imperatives has been changed to include an awareness of alcoholism and alcohol abuse as physiological aberrations whose etiology may be understood and for which therapy may be found through scientific pursuits. Both alcohol abuse and dependence arise as a result of different, complex, and as yet incompletely understood processes. At present, alcohol research is in the mainstream of scientific efforts.
Our studies on alcohol (ethanol or ethyl alcohol) have been based on the hypotesis that its abuse can ultimately be understood and dealt with at the molecular level. Such a molecular understanding, if achieved, would provide a basis for the identification and development of appropriate therapeutic agents. Our view hypothesizes that the clinical manifestations of alcoholism and alcohol abuse are the consequence of aberrations or defects within one or more metabolic pathways, affected by the presence of ethyl alcohol. In order to test this hypothesis, our initial studies focused on physical, chemical, and enzymatic properties of human alcohol dehydrogenase (ADH), the enzyme that catalyzes alcohol oxidation according to the following reaction formula: EQU CH.sub.3 CH.sub.2 OH+NAD+.fwdarw.CH.sub.3 CHO+NADH
In addition, our studies more recently have focused on the aldehyde dehydrogenases (ALDH) which catalyze the subsequent step in the major pathway of ethanol metabolism according to the following reaction formula: EQU CH.sub.3 CHO+NAD+.fwdarw.CH.sub.3 COOH+NADH
Prior to our research (for example, see Blair and Vallee, 1966, Biochemistry 5: 2026-2034), ADH in man was thought to exist in but one or two forms, primarily in the liver, where it was considered the exclusive enzyme for the metabolism of ethanol. Currently, four different classes of ADH encompassing over twenty ADH isozymes have been identified and isolated from human tissues. There is no reason to believe that all of these ADH isozymes are necessary to catalyze the metabolism of a single molecule, ethanol, even though all of them can interact with it. We have proposed that the normal function of these isozymes is to metabolize other types of alcohols that participate in critical, physiologically important processes, and that ethanol interferes with their function (Vallee, 1966, Therapeutic Notes 14: 71-74). Further, we predicted that individual differences in alcohol tolerance might well be based on both qualitative and quantitative differences in isozyme endowment (Vallee, 1966, supra).
Our research has established the structures, properties, tissue distribution, and developmental changes for most of the ADH isozymes, which while structurally quite similar, and presumed to have evolved from a common precursor, are functionally remarkably varied. Of the more than 120 publications from our laboratory that relate to the above subjects, the following, arranged in six categories, are especially useful for instruction in the prior art.
(i) Discovery of isozymes: Bosron et al., 1977, "Isolation and Characterization of an Anodic Form of Human Liver Alcohol Dehydrogenase," Biochem. Biophys. Res. Comm. 74: 85-91; Bosron et al., 1979, "Human Liver .pi.-Alcohol Dehydrogenase: Kinetic and Molecular Properties," Biochemistry 18: 1101-1105; Bosron et al., 1980, "New Molecular Forms of Human Liver Alcohol Dehydrogenase: Isolation and Characterization of ADH (Indianapolis)," Proc. Natl. Acad. Sci. USA 77: 5784-5788; Paris and Vallee, 1981, "New Human Liver Alcohol Dehydrogenase Forms with Unique Kinetic Characteristics," Biochem. Biophys. Res. Comm. 98. No. 1: 122-130.
(ii) Discovery of new physiological and toxicological substrates: Wacker et al., 1965, "Treatment of Ethylene Glycol Poisoning with Ethyl Alcohol," JAMA 194: 1231-1233; Frey and Vallee, 1980, "Digitalis. Metabolism and Human Liver Alcohol Dehydrogenase," Proc. Natl. Acad. Sci. USA 77: 924-927; M.ang.rdh et al., 1985, "Human Class I Alcohol Dehydrogenases Catalyze the Oxidation of Glycols in the Metabolism of Norepinephrine," Proc. Natl. Acad. Sci. USA 82: 4979-4982; M.ang.rdh et al., 1986a, "Testosterone Allosterically Regulates Ethanol Oxidation by Homo- and Heterodimeric .gamma.-Subunit-Containing Isozymes of Human Alcohol Dehydrogenase," Proc. Natl. Acad. Sci. USA 2836-2840; Consalvi et al., 1986, "Human Alcohol Dehydrogenases and Serotonin Metabolism," Biochem. Biophys. Res. Comm. 139: 1009-1016; Mardh and Vallee, 1986b, "Human Class I Alcohol Dehydrogenases Catalyze the Interconversion of Alcohols and Aldehydes in the Metabolism of Dopamine," Biochemistry 25: 7279-7282; Mardh et al., 1986c, "Human Class II (.pi.) Alcohol Dehydrogenase Has a Redox-Specific Function in Norepinephrine Metabolism," Proc. Natl. Acad. Sci. USA 83: 8908-8912; Mardh et al., 1987, "Thyroid Hormones Selectively Modulate Human Alcohol Dehydrogenase Isozyme Catalyzed Ethanol Oxidation," Biochemistry 26: 7585-7588; McEvily et al., 1988, "3.beta.-Hydroxy-5.beta.-steroid Dehydrogenase Activity of Human Liver Alcohol Dehydrogenase Is Specific to .gamma.-Subunits," Biochemistry 27: 4284-4288; Keung, 1991, "Human Liver Alcohol Dehydrogenases Catalyze the Oxidation of the Intermediary Alcohols of the Shunt Pathway of Mevalonate Metabolism," Biochem. Biophys. Res. Comm. 174: 701-707.
(iii) Development of new methods for isolation and characterization: Lange and Vallee, 1976, "Double-Ternary Complex Affinity Chromatography: Preparation of Alcohol Dehydrrogenases," Biochemistry 15: 4681-4686; Keung et al., 1985, "Identification of Human Alcohol Dehydrogenase Isozymes by Disc Polyacrylamide Gel Electrophoresis in 7M Urea," Biochem. Biophys. Res. Comm. 151: 92-96; Montavon et al., 1989, "A Human Liver Alcohol Dehydrogenase Enzyme-Linked Immunosorbent Assay Specific for Class I, II, and III Isozymes," Anal. Biochem. 176: 48-56.
(iv) Characterization of isozymes: von Wartburg et al., 1964, "Human Liver Alcohol Dehydrogenase. Kinetic and Physicochemical Properties," Biochemistry 3: 1775-1782; Blair and Vallee, 1966, supra; Lange et al., 1976, "Human Liver Alcohol Dehydrogenase: Purification, Composition, and Catalytic Features," Biochemistry 15: 4687-4693; Wagner et al., 1983, "Kinetic Properties of Human Alcohol Dehydrogenase: Oxidation of Alcohols by Class I Isoenzymes," Biochemistry 22: 1857-1863; Wagner et al., 1984, "Physical and Enzymatic Properties of a Class III Isozyme of Human Liver Alcohol Dehydrogenase: .chi.-ADH," Biochemistry 23: 2193-2199; Ditlow et al., 1984, "Physical and Enzymatic Properties of a Class II Alcohol Dehydrogenase Isozyme of Human Liver: .pi.-ADH," Biochemistry 23: 6363-6368; Fong and Keung(a), 1987, "Substrate Specificity of Human Class I Alcohol Dehydrogenase Homo-and Heterodimers Containing the .beta..sub.2 (Oriental) Subunits," Biochem. 26: 5726-5732; Fong and Keung(b), 1987, ".beta..sub.2 (Oriental) Human Liver Alcohol Dehydrogenases Do Not Exhibit Subunit Interaction: Oxidation of Cyclohexanol by Homo- and Heterodimers," Biochem. 26: 5733-5738.
(v) Relationship of isozymes to genetics: Li et al., 1977, "Isolation of Alcohol Dehydrogenase of Human Liver: Is it a Determinant of Alcoholism?," Proc. Natl. Acad. Sci. USA 74: 4378-4381; Jornvall et al., 1984, "Human Liver Alcohol Dehydrogenase: Amino Acid Substitution in the .beta..sub.2 .beta..sub.2 Oriental Isozyme Explains Functional Properties, Establishes an Active Site Structure, and Parallels Mutational Exchanges in the Yeast Enzyme," Proc. Natl. Acad. Sci. USA 3024-3028; von Bahr-Lindstrem et al., 1986, "cDNA and Protein Structure for the .alpha. Subunit of Human Liver Alcohol Dehydrogenase," Biochemistry 25: 2465-2470; Hoog et al., 1987, "Structure of the Class II Enzyme of Human Liver Alcohol Dehydrogenase: Combined cDNA and Protein Sequence Determination of the .pi.Subunit," Biochemistry 26: 1926-1932; Fong et al., 1989, "Liver Alcohol and Aldehyde Dehydrogenase Isozymes in a Chinese Population in Hong Kong," Human Heredity 39: 185-191.
(vi) Tissue distribution of isozymes: Pares et al., 1984, "Organ Specific Alcohol Metabolism: Placental .chi.-ADH," Biochem. Biophys. Res. Comm. 119: 1047-1055; Beisswenger et al., 1985, ".chi.-ADH is the Sole Alcohol Dehydrogenase Isozyme of Mammalian Brains: Implications and Inferences," Proc. Natl Acad. Sci. USA 82: 8369-8373.
One ADH isozyme, class III or .chi.-ADH, is the only one present in brain, placenta, and testis and is least capable of oxidizing ethanol (Pares and Vallee, 1981, supra Paris et al., 1984, supra; Beisswenger et al., 1985, supra). As a consequence, these tissues would seem to be at greatest risk with respect to the effects of ethanol. On the other hand, this circumstance also affords these tissues protection from acetaldehyde, the highly toxic oxidation product of ADH.
Alcohol abuse and alcoholism are problems unique to humans. It may not be surprising, therefore, that the complexity in other species is significantly less than in man. Such species differences extend to the catalytic preferences of ADH isozymes toward different alcohols. For example, horse ADH does not oxidize methyl alcohol and ethylene glycol while human ADH does (von Wartburg et al., 1964, supra). Large doses of ethanol administered to compete with methanol or ethylene glycol and prevent their oxidation to toxic products now constitutes the therapy for individuals poisoned with these agents (Wacker et al., 1965, supra). As a consequence of the detailed research exemplified above, much more is known about human ADH than the corresponding enzyme in other species, a unique situation quite the opposite for most other enzymes.
Each of the human ADHs is composed of two protein subunits that form a dimeric molecule. Class I ADHs are made up of .alpha., .beta., and .gamma. subunits which combine into homodimeric and heterodimeric isozymes; class II, III and IV appear to be only homodimers (Vallee and Bazzone, in Isozymes: Current Topics in Biological and Medical Research, Rattazzi et al. (eds.) pp. 219-244, Alan R. Liss, Inc., NY, 1983; Vallee, B. L., A Novel Approach to Human Ethanol Metabolism: Isoenzymes of Alcohol Dehydrooenase. Invited Lecture, Proceedings of the 20th International European Brewery Convention, Helsinki, 1985; Pares et al., 1990, FEBS Lett. 227: 115-118). The activities of the different ADHs toward several types of substrates has been examined and is quite revealing (see, for example, Vallee, 1985, supra). Class I isozymes containing at least one .gamma.-subunit are active toward specific steroid hormones and are selectively inhibited by testosterone (Mardh et al., 1986a, supra; McEvily et al., 1988, supra). Class II ADH contains the .pi.-subunit and is the only one that acts selectively on intermediates in the metabolism of norepinephrine, a critical endocrine and neurotransmitter agent (M.ang.rdh et al., 1986c, suora). The class III (.chi.) enzyme and its unique characteristics were mentioned above. The recently discovered human class IV ADH (Moreno and Paris, 1991, J. Biol. Chem., 266: 1128-1133), found mainly in gastric mucosa, shares the general physicochemical properties of all mammalian ADHs. Kinetically, it resembles class II ADH but is chemically distinct. Since ethanol concentration in the stomachs of drinkers may be as high as 1 to 10 M transiently, the moderately high K.sub.m, 41 mM, of this isozyme is nevertheless ample to allow it to have a possibly important role in the first pass metabolism of ethanol. Many alcohols other than ethanol have important physiological roles and some are likely to be substrates for one or another of the ADH isozymes. Clearly, the interference of ethanol with normal metabolic processes could have serious consequences, both acute and chronic. One of the main goals of continued research is the identification of these critical substrates.
Genetic subvariants of the .beta. and .gamma.-subunits of ADH isozymes within the general population (.beta..sub.1, .beta..sub.2, .beta..sub.3, and .gamma..sub.1, .gamma..sub.2) produce characteristic differences in individuals. The first genetic difference found between the form predominant in Caucasians (.beta..sub.1) and that predominant in Asians (.beta..sub.2) is also the most profound (Smith et al., 1971, Ann. Hum. Genet., Lond., 34: 251-271; Fukui and Wakasugi, 1972, Jpn. J. Leg. Med., 26: 46-51); the .beta..sub.1 -subunit is 100-times less effective in converting ethanol to acetaldehyde than is the .beta..sub.2 -subunit. All of the differences are now known to result from point mutations at widely different positions in the chain, e.g., .beta..sub.1.fwdarw..beta.2, R47H; .beta..sub.1.fwdarw..beta.3, R369C; .gamma..sub.1.fwdarw..gamma.2, I349V and R271Q but all affect coenzyme binding (Jornvall et al., 1987, Enzyme 37: 5-18). Several population studies have documented striking differences in .beta..sub.1 and .beta..sub.2 frequencies among Asian and Caucasian populations. For example, in an Asian population in Hong Kong, the .beta..sub.1 form of the .beta.-subunit was present in only about 10% of the subjects; all others had the .beta..sub.2 form (Fong et al., 1989, supra). In contrast, studies on a Caucasian population in England indicated that 90% had the .beta..sub.1 form and only 10% had the .beta..sub.2 form (Smith et al., 1971, supra).
Aldehyde dehydrogenase (ALDH) is the enzyme that catalyzes the second step in the ethanol metabolic pathway (see reaction formula above). As with ADH, there are multiple forms of ALDH, but only two of these have been examined in any detail; very much less is known about the others. The first two classes, in particular, are thought to have primary responsibility for oxidizing acetaldehyde (Pietruszko, in Biochemistry and Physiology of Substance Abuse, Watson (ed.), pp. 89-127, 1989). ALDH-I is present in mitochondria, has a high affinity for acetaldehyde, and has been assigned the major role in acetaldehyde detoxification. ALDH-II, on the other hand, occurs in the cytosol and has a low affinity for acetaldehyde. It is therefore thought to be less effective in its detoxification. The amino acid sequences of both forms are now known (Jornvall et al., 1987, supra).
An important inactive dominant mutant form of ALDH-I was discovered by Goedde et al., 1979, Hum. Genet. 51: 331-334, and shown to be present in approximately 50% of major Asian populations, e.g., Chinese, Japanese and Vietnamese (Goedde and Agarwal, 1987, Enzyme, 37: 29-44). This mutant protein apparently results from at least one point mutation (K487E) (Yoshida et al., 1984, Proc. Natl. Acad. Sci. USA 81: 258-261) that abolishes enzymatic activity and therefore markedly impairs the ability of heterozygous and homozygous individuals (Goedde and Agarwal, 1990, Pharm. Ther., 45: 345-371) to metabolize a variety of aldehydes including acetaldehyde and presumably including any physiologically important aldehydes that are in the range of the specificity characteristic of native ALDH-I. Remarkably, such individuals do not display any pathologic abnormalities but do experience a sensitivity reaction when they consume alcohol. The characteristic facial flushing is the symptom of this reaction that is recognizable immediately. Still more remarkably, this mutation seems to have survival value: alcoholism and alcohol abuse virtually do not exist among Asian flushers (Ohmori et al., 1986, Prog. Neuro-Psychopharmacol. and Biol. Psychiat., 10: 229-235).
The Hong Kong study (Fong et al., 1989, supra) documents, for the first time, the joint distribution of the .beta.-ADH and ALDH-I genetic subvariants in a Chinese population. The subvariants classify into four measurably distinct subgroups: 2.2% % .beta..sub.1 -ADH and active ALDH-I; 5.6% % .beta..sub.1 -ADH and inactive ALDH-I; 44.4% % .beta..sub.2 -ADH and active ALDH-I; and 47.8% .beta..sub.2 -ADH and inactive ALDH-I. Based on the catalytic capacities of the four phenotype varieties, one would expect subjects with .beta..sub.2 -ADH and inactive ALDH-I to be the most rapidly intolerant of alcohol; those with .beta..sub.1 -ADH and inactive ALDH-I to be intolerant of alcohol but with less rapid onset; those with .beta..sub.2 -ADH and active ALDH-I to be moderately tolerant; and subjects with .beta..sub.1 -ADH and active ALDH-I, i.e., the predominant Caucasian type, to be tolerant.
Since the lack of ALDH-I is not known to generate other significant metabolic problems, save those which are the consequence of ethanol metabolism, it would be ideal if a drug could be found which mimics the effect of this natural genetic variant but without producing substantial toxic side effects; such a drug would clearly offer great promise for the treatment of alcoholism and alcohol abuse.
The experience of Asian flushers with alcohol is not described as "aversion," but rather as intolerance. i.e., as an inability to endure alcohol. This is an important distinction because in Western medicine the psychological setting surrounding the administration of the toxic drugs disulfiram and carbimide has been given considerable emphasis in producing a regimen leading to so-called "aversion therapy" and more recently "psychological deterrence " (Banys, 1988, J. Psychoactive Drugs 26: 243-261).
We now describe in detail two types of treatments for alcoholism and alcohol abuse that were known long before either the enzymology or genetics of ADH and ALDH isozymes were known. Their discovery and use has been phenomenological: not based on modern rational drug discovery or design. On the one hand, Western medicine has used toxic chemicals, not further developed since discovery of their effects on exposed industrial workers decades ago, to produce sensitization to alcohol. Ancient Traditional Chinese medicine, on the other hand, has used herbal preparations to treat diseases generally, and in particular alcohol intoxication, according to a philosophy in which herbal mixtures modulate bodily functions; treatment with herbal combinations is highly individualistic both with respect to the practitioner's preferences and prescriptions for the patient; record-keeping is rare; and practice of the art is heavily influenced by oral anecdotal tradition.
The only two pharmaceuticals currently used as alcohol-sensitizing drugs are both chemically reactive species but differently so, both non-specific inhibitors and individually distinct and hence different from one another, and both shown after decades of testing and use to be toxic, unsafe and ineffective. The pharmacological basis for the action of these drugs, disulfiram and carbimide (hereinafter referred to by its chemical name, cyanamide) is thought to be inhibition of hepatic ALDHs, but neither one is specific for ALDH-I, the only ALDH known to be affected by genetic mutation.